Microbial tests from chronic wounds
If we suspect an infection in the wound and consider starting a course with antibiotic treatment, we should try to find out which bacteria might be causing the infection. We are left with two choices besides advanced techniques like PCR analysis, which is only found at very few African institutions. We can either do a bacterial swab and transfer the swab onto a culture plate to multiply the bacteria in a few days. Once the bacteria have multiplied, they are transferred to a glass plate, stained ( usually with Gram stain), and examined under a microscope. The other option is to take a sample from the wound and transfer this directly to a glass plate. A Gram stain or Ziehl-Nielsen stain ( for acid-fast bacilli) should be done before examination under the microscope. The advantage of the latter method is that it is cheap and quick - within about 20 minutes, you may already have examined the sample. The disadvantage is that if there are few bacteria, you may miss them. Also, a direct examination will carry along cell debris and other artifacts, making visualization of the bacteria more difficult. Furthermore, you will not be able to analyze bacterial resistance. When using the culture plate technique, we can place small bits of antibiotic-laden paper onto the culture to check which antibiotics have the best effect on that specific strain of bacteria.
When we look for acid-fast bacilli like Mycobacterium ulcerans ( Buruli ulcer), a direct stain from the swab is best because it will take many weeks before you, even under optimal conditions, can get them to grow in a culture. These types of bacteria grow very slowly. A PCR test is even better for identifying mycobacteria, but as we already stated, this type of test is most likely not available to you.
Figure 1. In general, there are two methods to check which bacteria may be causing an infection in the wound. The first method ( A) transfers the swab to a growth medium ( often blood agar) to multiply the bacteria first. In this way, we get a higher concentration of bacteria, which makes identifying the strain much easier. The downside is that the culture process usually takes 2-3 days. After the bacteria have been cultured, a regular stain and examination under the microscope is done.
The direct method (B) involves taking a sample from the wound bed and transferring this directly to a glass slide, staining it, and examining the slide immediately. Because the bacterial numbers are lower, sometimes they can be harder to identify. Usually, a Gram stain or a Ziehl-Nielsen stain is done depending on what bacteria you suspect are the culprits.
Figure 2 Another advantage of culturing bacteria on a growth medium is that we can test how different antibiotics perform on that particular strain. For this purpose, small round paper plates saturated with different antibiotics are laid on the culture, and then we observe how close to these antibiotics the bacteria grow. The image above shows that the bacteria completely resist the antibiotic with the blue arrow. Here, we see bacteria growing right up to the paper containing that particular antibiotic. This is beneficial information when choosing an antibiotic for treating the patient.
When should you do a bacterial swab from a chronic wound?
There are far too many bacterial swabs taken from chronic wounds! This has led to an overuse of antibiotics in chronic wounds worldwide. It is essential to understand that you ALWAYS will grow some bacteria from a chronic wound. It is, for example, very usual to find Staphylococcus aureus in many chronic ulcers. It is also not unusual to find pseudomonas aeroginosa, e-coli, or other enterococci in chronic wounds. This does not mean that the patient needs antibiotics! It is completely wrong to think. "let's do a bacterial swab and see what turns up - if we find pathological bacteria, we will start with a course of antibiotics."
Figure 3 Remember that you always will find bacteria in a chronic wound in the same way you always will find bacteria living on your skin. If you take a routine swab from your skin, you will most likely get a nice mixture of potentially pathological bacteria, although you are perfectly healthy. You would not place a person on a course of antibiotics only because you found staphylococcus aureus in a swab from the skin, right?
As a rule of thumb: if you are thinking of starting a patient with a chronic ulcer on an antibiotic course, you should do a bacterial swab. Once the swab has been performed, you can begin the antibiotic treatment. As soon as the test results are back, you must check if the antibiotics you have administered are the right type for the bacterial strains shown. If you have a good laboratory at hand, they will have done a resistance test to show which antibiotics are most advisable in this specific case.
The question, however, is - should you start the patient on antibiotics at all? If there are clear signs of a significant infection or the patient even has systemic signs of infection, then antibiotic treatment is most likely necessary. Just having some redness around a chronic ulcer is by no means a sure sign of infection - in most cases, this is simply the body's inflammatory response to the fact that there is a hole in the skin at this place.
At the tissue viability center where we work, we very rarely use antibiotics, even though we often deal with large and complicated ulcers. We never never do a routine bacterial swab of an ulcer just to be curious about who is living at the bottom of the ulcer.
However, in diabetic patients with deep foot ulcers, we have a low threshold for starting with antibiotic treatment, especially if the ulcer is close to the bone- or tendon structures. These ulcers quickly can deteriorate, and we do not always see the typical infection signs of redness, swelling, and pain in diabetic patients. In other words- in more challenging diabetic feet ulcers, we routinely take bacterial swabs because we often use antibiotics here.
Another situation where we might take a bacterial swab, although the wound is not clearly infected, is when we have an ulcer that is not healing as expected. In such cases, you should also take a tissue biopsy for histological analysis, but you may also consider a bacterial swab at the same time. Sometimes colonization with, for example, streptococci or pseudomonas can be invisible to the eye but impair healing to such an extent that the wound just stalls. This doesn't mean that the presence of streptococci or pseudomonas in a wound always means trouble - but in some cases, it does. Suppose you believe that the problem with the wound is unfortunate colonization with a strain of bacteria. In that case, you should do a swab and first try a topical antibacterial dressing to eliminate the bacteria in this way. There is a wide choice of antimicrobial dressings available - ranging from silver-coated dressings and iodine dressings to honey- and sugar preparations.
How should you do a microbial swab?
Our standard swab for bacterial specimen collection and transportation is a black-capped swab containing Amies gel medium with charcoal. It is intended to recover aerobic, anaerobic, and fastidious bacteria. The charcoal in the medium neutralizes substances that are toxic to sensitive bacteria. All swabs should be CE marked and meet the Clinical Laboratory Standard Institute approved standard M40-A2. If you can only get hold of non- CE marked swabs, you have to use these and hope that they meet basic standards.
Figure 4 Amies medium is the standard swab for bacterial cultures from chronic ulcers. Remember that the swab is sterile and that it is important not to contaminate the swab while you take the sample. In other words, it is essential that you only come into contact with the desired area in the wound and are careful not to touch the peri-wound area, the patient's clothes, or your gloved fingers with the swab tip.
A chronic ulcer usually contains a mixture of different bacteria. When we are dealing with an infection of the wound, only rarely are all bacteria involved in the infection. In most cases, only one of the bacterial strains is the real culprit. These are usually the bacteria left behind when we have rinsed the wound thoroughly. So - to find the real culprits, we have to wash away all the unimportant bystanders and expose those who are remaining. To do this, we usually have to debride the wound well and then rinse it thoroughly. In our courses, we are often asked whether this won't wash away all the bacteria. Don't worry - you will never manage to wash away all the bacteria, and you can be quite sure that the relevant bacteria still will be hiding on the surface of the wound bed, ready to be detected with your swab. The more you rinse the wound bed, the more exact your swab!
Only use sterile saline when rinsing the wound before taking a swab. Ideally, the rinsing solution should be around body temperature. This is because cold rinsing solutions may make the bacteria less vital. Besides, the wound itself prefers not to be cooled down. Do not use antimicrobial rinsing solutions like polyhexanide (prontosan), super oxidized water, or vinegar-based rinses, as these may give you a false negative bacterial swab!
When you take the swab, press or rub the tip of the swab gently over the deepest portion of the wound bed - this is usually the best catchment area. When you have areas of exposed bone or tendon, take the bacterial samples from these tissues. If the surface is dry, then moistening the tip of the swab with sterile saline will help bacteria to adhere to the swab. Sample the entire surface of the area suspected to be infected using a zig-zag motion while simultaneously rotating the swab between the fingers. Place the swab into the transport medium immediately after swabbing. Be very careful not to contaminate the tip of the swab as you retreat it out of the wound.
Filling out the microbiology request for the lab
Remember to write the patient's name on the outside of the swab cover and on the request form to the lab! This should be unnecessary to mention but is surprisingly often forgotten. A bacterial swab without a name is useless and is usually discarded.
The microbiologist depends on much clinical information to make a sensible decision about which bacteria in the culture are most relevant. The microbiologist will find it helpful to know which kind of ulcer the patient has. In a venous leg ulcer, for example, usually, only one bacteria is the cause of the infection. In a diabetic foot ulcer, more often, two ( or more) bacteria may be actively involved in the infection. The laboratory also needs information about where the ulcer is located - do not just write "pressure ulcer" on the request. If you write "sacral pressure ulcer," the microbiologist will understand why there are many e-coli and enterococcus bacteria in the swab. After all, the sacrum is near the anus, and it is common to find intestinal bacteria in sacral pressure ulcers. You must write whether the patient has been using antibiotics recently and whether antimicrobial rinsing solutions or antimicrobial dressings ( silver, iodine, honey, sugar?) have been used. It is also essential to write that your swab has been taken from a cleaned ( and debrided) wound bed.
If you have taken a swab from a wound where you suspect Mycobacterium ulcerans (Buruli ulcer) to be present, it is imperative that you clearly convey this to the laboratory. This type of bacteria requires other staining and culturing methods!
Storing the swab
A bacterial swab is a fresh product and should be sent to the lab as soon as possible. If you cannot send it the same day because of logistical reasons, you should store the swab in a fridge at a temperature between 4- 8 ° C until the next day. Be aware that the sample may not be as good the next day - you may not get the growth of all bacteria. The best is to send the swab to the lab the same day!
What can you do if you are working off the grid with no access to a laboratory?
In most areas in Africa, even in quite remote areas, there is usually a clinic or dispensary where they may be someone with a microscope. Whether they can do a basic bacterial culture analysis is another question. These health workers often have some training in analyzing blood slides to test for malaria, for example, which only requires basic staining techniques. Doing a bacterial culture with culture plates which have to be incubated etc., is another thing. However, you will find that if they can do a malaria stain, they will also be able to do a Gram stain for bacterial analysis.
Remember that you do not necessarily have to culture the bacteria before analyzing them. You can take a swab directly from the wound and smear the swab directly onto a glass plate and stain this with a gram stain. The problem with this "direct" technique is that you will see a lot of artifacts ( cell debris etc.) and only a few bacteria as they have not been concentrated by culture on an agar plate. With a bit of training, however, you will be able to recognize at least the most common pathogenic bacteria.
The Gram stain provides preliminary results on whether bacteria are present ( they will always be present in a chronic wound!) and the general type, such as the shape and whether they are Gram-positive or Gram-negative. This will better indicate which antibiotic to use rather than just taking an educated guess by looking at the infected wound.
Using this direct method, you can use a clean cotton q-tip to transfer the bacteria from the wound bed onto the glass slide. Clean and rinse the wound thoroughly as you would do when doing a swab before taking your sample.
Again, this is great if you have access to a modern microbiological laboratory that can culture the swab to concentrate the bacteria and even do antibiotic resistance tests; if you do not have access to this type of service, you will have to manage by doing a direct gram stain.
Figure 5 With some basic training, it is not difficult to do a gram stain and at least identify the most common pathogenic bacteria. The disadvantage of this technique, as opposed to culturing the bacteria first, is that there often are many artifacts in the slide, and the number of bacteria is usually relatively low, making the interpretation more difficult.
Video 1 A short presentation on how to do a gram stain. You will need a heat source, three types of dyes, and alcohol- all these items are very cheap and usually available from various suppliers in Africa. The staining technique is straightforward to learn - the interpretation of the slides under the microscope requires some more training.
How do you interpret the microbiological results?
When working at a clinic where you regularly send bacterial swabs to a laboratory, you have to have a system to check the results as soon as they return and check which antibiotic treatment the patient has received. It should seem obvious that this should be done - but from experience, at our clinic, we regularly see that someone forgets to check whether the antibiotic prescribed is adequate for the bacteria found in the culture. For example, suppose we prescribed a beta-lactam antibiotic to a patient with a wound infection, and the bacterial swabs primarily show enterococci growth. In that case, we should switch to a different antibiotic. You have to establish a routine for this and determine who is responsible for phoning the patient and informing them about the need to change antibiotics. If you have a laboratory that performs antibiotic resistance tests, you can tailor the antibiotic treatment even more accurately.
Even if we have cleaned and rinsed the wound thoroughly before taking the sample, we may have a mixture of bacteria in the lab report. This will make the choice of antibiotics more difficult. Remember- it is pretty rare to have several bacteria equally involved in causing an infection. Usually, only one strain of bacteria found in the culture is responsible for the infection. Instead of prescribing broad-spectrum antibiotics, we should use our clinical experience to make an educated guess about who is the true culprit and use narrow-spectrum antibiotics as often as possible. The smell of the infected wound can give us important clues - does the wound smell rotten or even feces like - then most often, some intestinal bacteria is the culprit of the infection. Staphylococcal infections do not usually make smelly wounds. If the skin around the wound is intensely red and the redness is spreading quite rapidly, you should suspect streptococci or staphylococci to be the culprits. If there is greenish exudate in the wound, this indicates infection with pseudomonas.
If you have debrided the wound well and rinsed it thoroughly, you will most often get a much more exact lab result - if you are lucky, the results will only show one strain of bacteria, and the choice of antibiotic is much easier.
Figure 6 Results from a bacterial swab from a chronic wound cleaned well and rinsed thoroughly before taking the swab. The results show only staphylococcus aureus growing in the culture, and the antibiotic resistance testing showed resistance to erythromycin and clindamycin. Note: the lab report is in the Norwegian language, but the essential part of the text is universally understandable. The letters "S" And "R" behind each antibiotic stand for sensitive and resistant, respectively.
Figure 7 An example of a poorly performed swab from a chronic wound. The wound was not cleaned and appropriately rinsed before the sample was taken, and the test results will give you a headache. The microbiologists found six different strains of bacteria growing in the culture - each of the strains were equally abundant! We can assure you that not all six strains were responsible for the patient's infection in the wound. And trying to interpret the antibiotic resistance test is undoubtedly a challenge - which antibiotic should we use here??? This bacterial swab was a waste of time, and we should definitely not prescribe three different antibiotics to cover all six strains of bacteria! We hope this example clearly demonstrates the problems you will face if you do not clean and rinse the wound thoroughly before doing a swab! Note: the lab report is in the Norwegian language, but the essential part of the text is universally understandable. The letters "S" And "R" behind each antibiotic stand for sensitive and resistant, respectively.
How do you take a bacterial swab from the bone where you suspect osteomyelitis?
Osteomyelitis is an infection of the bone. The treatment necessitates many weeks, sometimes months, of antibiotic therapy. It is, therefore, crucial that we are as sure as can be that we have chosen the right antibiotic for the bacteria that are causing the bone infection. It is simply quite tragic if you treat the patient for 12 weeks with an antibiotic only to discover that the treatment is not effective because you chose the wrong antibiotic.
Therefore, if you are treating osteomyelitis, it is essential to have secured a bacterial analysis before starting the antibiotic treatment. If a patient already has been taking some antibiotics without a swab having been taken, you should pause the antibiotic therapy for 10-14 days and then do the bacterial swab.
Even if you have an open ulcer above the infected bone - it is not very helpful to just take a bacterial swab from the wound - this will not necessarily tell you which bacteria is living in the bone. Essentially you have to take a bacterial swab from within the bone.
It is not a good idea to go through the open wound to take the swab from the bone as you almost always will touch the wound bed with the swab as you enter the bone - the swab will get contaminated with maybe completely irrelevant bacteria. Sometimes, however, there is a large visible bone defect in the open wound, and it may be possible to enter the bone with the swab without touching the wound bed.
The gold standard method is to enter the bone through a separate incision in an area unaffected by the wound, use a surgical tool to open the bone and insert the swab there. This will give you the best test results without any danger of wound contamination. This demands training and resources which are not available everywhere. If there is deep osteomyelitis of the pelvis, sometimes CT- or MRI-guided techniques are used at high-level medical centers to take precise bacterial tests from deeper within the body.
Even though we have the described gold standard above, many wound practitioners settle for taking a bacterial swab through the open wound because this is more practical and easy to perform. If you clean the wound very thoroughly and rinse it very well beforehand - in this situation, you could even use antibacterial rise solutions like super oxidized water or 3,5% vinegar - and a very careful not to touch the wound bed with the swab, you can get a reasonably accurate sample. At our clinic, we often use this technique.
If the surface of the bone is necrotic, you can be very sure that there is osteomyelitis lurking beneath, and you can use a ring curette to remove the upper millimeters of the necrotic bone and take a swab from beneath here.
Cultures from bone swabs can be disappointing, and a tip to get more consistent results is to scrape away some of the rotten bone - place this in a sterile syringe closed with a Luer lock. Fill the syringe with about 6-8 ml of sterile saline, and after replacing the plunger, shake the syringe vigorously for about a minute to wash the bacteria out of the bone. The resulting broth is injected equally into blood culture mediums ( anaerobic and aerobic) and sent for culturing to a laboratory. We have more often succeeded in isolating the bacteria causing osteomyelitis with this method. You will obviously need a blood culture medium available for this and preferably a laboratory that can do the analysis. If you do not have such a laboratory, then you can place the blood culture mediums in a cabinet at 37 degrees centigrade and let the bacteria multiply for 12-24 hours. After the first 12 hours, you can aspirate a few milliliters of each glass and do a gram stain from each glass. If you do not see enough bacteria to make a definite diagnosis wait for another twelve hours and repeat the stains.
If you have exposed bone in the wound, but the bone is white and healthy-looking, you must think twice about creating a defect in the bone to take a bacterial sample!!! Don`t do it is what we are saying! If you have made a hole in the bone, bacteria from the wound will definitely get into the bone and cause osteomyelitis. You should only open the bone when you are 100% sure that there is osteomyelitis lurking beneath - that is, either you can clearly see the necrotic bone at the bottom of the wound with your eyes, or you have the diagnosis confirmed by an x-ray, CT scan or even MRI-scan.
Figure 8 The image above shows a bone biopsy probe inserted into the distal phalange of a toe under CT scan guidance. You can clearly see changes in the bone indicative of osteomyelitis (messy appearance of the bone structure). The ulcer was on the plantar side of the toe, and the probe was passed into the bone in an area with intact skin so as not to contaminate the probe with irrelevant bacteria from the wound. This is the gold standard for taking a microbial biopsy from an osteomyelitis site. The procedure can also be done using regular x-ray or fluoroscopy. The procedure can also be done without radiologic imaging support in easily accessed areas like the toes.
Figure 9 We prefer blood culture mediums to culture bacteria when we take microbial samples from bone. As we described above, this involves scraping some necrotic bone from the site of the osteomyelitis, placing this in a sterile container with about 6-8 ml of sterile saline, and shaking this well for about a minute to wash bacteria out of the bone. The broth is injected into the culture mediums and incubated for 12-14 hours, sometimes longer. After this, we do gram stains from the mediums or get the cultures analyzed in a laboratory. This method has given us consistent, reliable results from osteomyelitis samples where the bacterial counts may be lower than in other infected tissue.
What about checking for fungal infections?
In wound care, fungal infections are often overseen. We are usually much more concerned about bacterial infections. And indeed, without a doubt are, bacterial infections what we encounter most.
Most commonly, we encounter fungal infections in the toenails of our patients, and often we ignore this as the ulcers on the legs and feet demand our attention more. Note that sometimes an infected toenail can give rise to an infection of the nailbed, and this is often overseen as the discolored toenail hides from what is underneath. This sort of situation is not rare in diabetic patients.
Another common site for fungal infections is the moist area between toes. While this condition, commonly known as athlete's foot, can cause itching and mild discomfort in healthy individuals, it can cause limb-threatening secondary infections in diabetic patients and those with a reduced immune system.
Another quite common situation is fungal infections arising in moist skin folds, such as under the breasts, groin areas, or abdominal folds in overweight patients. These fungal infections can be aggressive, causing extensive ulcerations and secondary infections.
In Africa, where we more often encounter HIV patients, fungal infections of the skin or even systemic fungal infections are something we must always keep in mind.
Be aware, however, that opportunistic fungus also can colonize a chronic wound and cause a wound to stall without making any big fuss out of it- in other words, there are no, if only a few, tell-tale signs of a fungal infection. We recall a patient with poor arterial circulation and small ulcers with sinus tracts on the toes. There were no inflammation signs but drops of light greyish purulent exudate coming from the sinus tracts for many weeks. Repeated bacterial analysis showed no growth of bacteria. After a few months, we did a fungal test that showed abundant fungal growth. The patient rinsed the sinus tracts daily with a vinegar-zinc solution, and the sinus tracts healed within a few weeks. Our theory is that previous repeated antibiotic treatments gave rise to an opportunistic fungus that saw his chance of colonizing the sinuses when the bacteria had moved out. This story shows that sometimes it is very useful to keep in mind that fungus infections also can give us problems in chronic wounds, and ideally, we should be able to test for these.
Another advantage of culturing fungal samples is that it is possible to do resistance tests for antimycotic treatments in the same way we test bacterial cultures for antibiotic resistance.
So how do we go about doing a test to check for fungi? Fungi are not as easily cultivated as bacteria - at least the process takes longer. If you have fungal lesions on relatively intact skin, you can take skin scrapings and examine these under the microscope. If you take a sample from a chronic ulcer or a wound sinus, you will most likely not see anything useful with direct microscopy, and a fungal culture is indicated. You can use the same type of swab as for bacterial cultures- Amies medium - and send this to a laboratory for culture.
The most reliable method for testing for fungi is PCR analysis. PCR stands for polymerase chain reaction. This is an expensive but ingenious method for detecting even small quantities of fungal ( or other microbial particles). In most regions in Africa, this is only available at large clinics, but we expect that more and more private clinics in Africa will invest in this technology, making it gradually more available, at least in large urban areas. Due to the Covid-19 pandemic, PCR machines have been more readily available on the African continent. While these machines are currently reserved for viral diagnostics, it is expected that these will be available for other microbiological tests in the future.
The Aga Khan University in Karachi, Pakistan, has written a very informative up-to-date manual for diagnosing fungal infections and made this available worldwide at ecommons.aku.de. Please refer to this manual for more information on this topic- click on the image below to get to a pdf version of the document.
